RESUMO
In a previous study, we detected a 6p25-p24 region linked to schizophrenia in families with high composite cognitive deficit (CD) scores, a quantitative trait integrating multiple cognitive measures. Association mapping of a 10 Mb interval identified a 260 kb region with a cluster of single-nucleotide polymorphisms (SNPs) significantly associated with CD scores and memory performance. The region contains two colocalising genes, LYRM4 and FARS2, both encoding mitochondrial proteins. The two tagging SNPs with strongest evidence of association were located around the overlapping putative promoters, with rs2224391 predicted to alter a transcription factor binding site (TFBS). Sequencing the promoter region identified 22 SNPs, many predicted to affect TFBSs, in a tight linkage disequilibrium block. Luciferase reporter assays confirmed promoter activity in the predicted promoter region, and demonstrated marked downregulation of expression in the LYRM4 direction under the haplotype comprising the minor alleles of promoter SNPs, which however is not driven by rs2224391. Experimental evidence from LYRM4 expression in lymphoblasts, gel-shift assays and modelling of DNA breathing dynamics pointed to two adjacent promoter SNPs, rs7752203-rs4141761, as the functional variants affecting expression. Their C-G alleles were associated with higher transcriptional activity and preferential binding of nuclear proteins, whereas the G-A combination had opposite effects and was associated with poor memory and high CD scores. LYRM4 is a eukaryote-specific component of the mitochondrial biogenesis of Fe-S clusters, essential cofactors in multiple processes, including oxidative phosphorylation. LYRM4 downregulation may be one of the mechanisms involved in inefficient oxidative phosphorylation and oxidative stress, increasingly recognised as contributors to schizophrenia pathogenesis.
Assuntos
Transtornos Cognitivos/genética , Homologia de Genes/genética , Proteínas Reguladoras de Ferro/genética , Proteínas Mitocondriais/genética , Regiões Promotoras Genéticas/genética , Esquizofrenia/genética , Psicologia do Esquizofrênico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Casos e Controles , Linhagem Celular , Transtornos Cognitivos/complicações , Feminino , Expressão Gênica/genética , Estudos de Associação Genética/estatística & dados numéricos , Humanos , Proteínas Reguladoras de Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/metabolismo , Fenilalanina-tRNA Ligase/genética , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/complicaçõesRESUMO
We consider the influence of a terahertz field on the breathing dynamics of double-stranded DNA. We model the spontaneous formation of spatially localized openings of a damped and driven DNA chain, and find that linear instabilities lead to dynamic dimerization, while true local strand separations require a threshold amplitude mechanism. Based on our results we argue that a specific terahertz radiation exposure may significantly affect the natural dynamics of DNA, and thereby influence intricate molecular processes involved in gene expression and DNA replication.
RESUMO
Xenograft outcomes are dictated by xenoantigen expression, for example, Gal alpha1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal-transferase gene-knockout (GalT-KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis-related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF-transgenic and GalT-KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal-expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT-KO grafts (1 of 14). Protective-type genes (heme oxygenase-I, superoxide dismutases and CD39) together with von Willebrand factor and P-selectin were upregulated in all renal grafts. Transcriptional responses in Gal-expressing xenografts were comparable to those seen in the infrequent GalT-KO rejection. In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor-1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Transplante de Rim/imunologia , Transplante Heterólogo/imunologia , Sistema ABO de Grupos Sanguíneos/imunologia , Doença Aguda , Animais , DNA Complementar/genética , Rejeição de Enxerto/mortalidade , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração/mortalidade , Transplante de Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Papio , Proteínas/genética , Suínos/genética , Timo/transplante , Condicionamento Pré-Transplante/métodosRESUMO
The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenovírus Humanos/genética , Sequência de Bases , Primers do DNA/química , Proteínas de Ligação a DNA/química , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , TATA Box , Proteína de Ligação a TATA-Box , Fatores de Transcrição/química , Transcrição Gênica , Proteína Supressora de Tumor p53/químicaRESUMO
Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed beta-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/biossíntese , Miocárdio/citologia , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Western Blotting , Bromodesoxiuridina/metabolismo , Ciclo Celular/genética , Divisão Celular , Núcleo Celular/química , Quinase 2 Dependente de Ciclina , DNA/análise , DNA/biossíntese , Expressão Gênica , Camundongos , Camundongos Transgênicos , Modelos Animais , Pressão , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
It has been suggested that thermally induced separations ("bubbles") of the DNA double-strand may play a role in the initiation of gene transcription, and an accurate understanding of the sequence dependence of thermal strand separation is therefore desirable. Based on the Peyrard-Bishop-Dauxois model, we show here that the bubble forming ability of DNA can be quantified in terms of a healing length L(n), defined as the length (number of base-pairs) over which a base-pair defect affects bubbles involving n consecutive base-pairs. The probability for a bubble of size n is demonstrated to be proportional to the number of adenine-thymine base-pairs found within this length. The method for calculating bubble probabilities in a given sequence derived from this notion requires several order of magnitude less numerical effort than direct evaluation.
Assuntos
Pareamento de Bases , Reparo do DNA , DNA/química , Modelos Químicos , Modelos Moleculares , Análise de Sequência de DNA/métodos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido NucleicoRESUMO
The acetylation and auto-acetylation of general transcription factors has recently been demonstrated, but the functional significance of these modifications is unclear. The presence of acetyl-coenzyme A activates basal transcription, and acetylation of transcription factor IIB (TFIIB) has been shown to activate transcription in several contexts. If auto-acetylation is an important pathway in eukaryotes, the regulatory pathways for acetyl-coenzyme A should be important in transcription regulation. Fasting represents an acute metabolic stress which should elevate levels of acetyl-coenzyme A, while mitochondrial aging represents a cumulative stress. We show that tissue-specific levels of acetylated TFIIB change dramatically in response to fasting in mice, suggesting a role for metabolism in the direct regulation of transcription. We also observed a large increase in acetyl-TFIIB in tissues from aged mice relative to younger mice. Sir2 family deacetylases, which regulate acetyl-coenzyme A synthesis, have recently been shown to impart longevity in a variety of organisms through a pathway related to calorie restriction. We hypothesize that protein acetylation and Sir2-related deacetylation may be tied to the metabolic regulation of transcription through the availability and action of acetyl-coenzyme A on key transcription factors and transcriptional regulators.
Assuntos
Acetilcoenzima A/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição TFIIB/metabolismo , Transcrição Gênica , Acetilação , Envelhecimento/fisiologia , Animais , Restrição Calórica , CamundongosRESUMO
The Ying-Yang 1 protein (YY1) DNA-binding site functions as an initiator element at which YY1, transcription factor IIB (TFIIB), and RNA polymerase II sponsor basal transcription from a supercoiled DNA template. We show that TFIIB binds to YY1, stabilizing its interaction with DNA, and YY1 contacts the large subunit of polymerase II, directing it to the initiation site. YY1 directs initiation from linear DNA containing mismatched sequences within its binding site, leading us to infer that supercoiling facilitates the separation of DNA strands and to suggest that YY1 likely remains bound to the start site as DNA strands separate during initiation. These results provide a mechanistic basis for transcriptional initiation directed by YY1 in the absence of the TATA box-binding protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/química , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Células HeLa , Humanos , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TATA Box , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Transcrição Gênica , Fator de Transcrição YY1RESUMO
YY1 is a zinc finger transcription factor whose DNA-binding motif exhibits the properties of an initiator element. Only three factors were required to direct specific basal transcription on a supercoiled template DNA carrying the YY1 initiator: YY1, general transcription factor IIB, and RNA polymerase II. This minimal in vitro reaction did not require the TATA-binding protein (TBP). We propose that, under appropriate conditions, YY1 can function like TBP, as a factor that binds to the core promoter and recruits the polymerase to the initiation complex.
Assuntos
DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/fisiologia , RNA Polimerase II/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Adenoviridae/genética , Sequência de Bases , DNA Viral/genética , Dependovirus/genética , Fatores de Ligação de DNA Eritroide Específicos , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Proteína de Ligação a TATA-Box , Moldes Genéticos , Fator de Transcrição TFIIB , Fator de Transcrição YY1RESUMO
Specialized proteins known as molecular chaperones bind transiently to non-native conformational states of proteins and protein complexes to promote transition to a biologically active conformation. Recently, it was demonstrated in vitro that proteins do not uniquely possess this activity. We show that mitochondrial 12S and 16S ribosomal RNA can fold chemically denatured proteins and reactivate heat-induced aggregated proteins in vitro. This chaperone action is ATP-independent. The specific secondary structure of the mitochondrial rRNA is critical to its folding activity. Furthermore, mutant mitochondrial 16S rRNA from aged cardiac muscle cells lacked this activity. We propose that mitochondrial 12S and 16S ribosomal RNA may play an important role in protein folding in mitochondria.
Assuntos
Desoxirribonuclease EcoRI/metabolismo , Enzimas/metabolismo , Dobramento de Proteína , RNA Ribossômico 16S/metabolismo , RNA Ribossômico/metabolismo , RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Besouros , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonuclease EcoRI/química , Enzimas/química , Humanos , Cinética , Luciferases/química , Luciferases/metabolismo , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Camundongos , Mitocôndrias Cardíacas/enzimologia , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Desnaturação Proteica , RNA/química , RNA Mitocondrial , RNA Ribossômico/química , RNA Ribossômico 16S/química , Suínos , Transcrição Gênica , Proteínas ViraisRESUMO
Ying Yang 1 (YY1) is shown to bind to the proximal promoters of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, farnesyl diphosphate (FPP) synthase, and the low density lipoprotein (LDL) receptor. To investigate the potential effect of YY1 on the expression of SREBP-responsive genes, HepG2 cells were transiently transfected with luciferase reporter constructs under the control of promoters derived from either HMG-CoA synthase, FPP synthase, or the LDL receptor genes. The luciferase activity of each construct increased when HepG2 cells were incubated in lipid-depleted media or when the cells were cotransfected with a plasmid encoding mature sterol regulatory element-binding protein (SREBP)-1a. In each case, the increase in luciferase activity was attenuated by coexpression of wild-type YY1 but not by coexpression of mutant YY1 proteins that are known to be defective in either DNA binding or in modulating transcription of other known YY1-responsive genes. In contrast, incubation of cells in lipid-depleted media resulted in induction of an HMG-CoA reductase promoter-luciferase construct by a process that was unaffected by coexpression of wild-type YY1. Electromobility shift assays were used to demonstrate that the proximal promoters of the HMG-CoA synthase, FPP synthase, and the LDL receptor contain YY1 binding sites and that YY1 displaced nuclear factor Y from the promoter of the HMG-CoA synthase gene. We conclude that YY1 inhibits the transcription of specific SREBP-dependent genes and that, in the case of the HMG-CoA synthase gene, this involves displacement of nuclear factor Y from the promoter. We hypothesize that YY1 plays a regulatory role in the transcriptional regulation of specific SREBP-responsive genes.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica , Dedos de Zinco , Alquil e Aril Transferases/metabolismo , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Geraniltranstransferase , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Regiões Promotoras Genéticas , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fator de Transcrição YY1RESUMO
Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II resulted in the loss of transcriptional activity. Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect. Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column. Fractionation of RNA polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA polymerase II. The phosphorylated form of the enzyme was unable to interact with TBP. The specific interaction of RNA polymerase II with TBP was mediated by the CTD of RNA polymerase II.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Extratos Celulares , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Proteína de Ligação a TATA-Box , Temperatura , Fator de Transcrição TFIIDRESUMO
Ying-Yang 1 protein (YY1) supports specific, unidirectional initiation of messenger RNA production by RNA polymerase II from two adjacent start sites in the adeno-associated virus P5 promoter, a process which is independent of the TATA box-binding protein (TBP). The 2.5-A resolution YY1-initiator element cocrystal structure reveals four zinc fingers recognizing a YY1-binding consensus sequence. Upstream of the transcription start sites protein-DNA contacts involve both strands and downstream they are virtually restricted to the template strand, permitting access to the active center of RNA polymerase II and ensuring specificity and directionality. The observed pattern of protein-DNA contacts also explains YY1 binding to a preformed transcription bubble, and YY1 binding to a DNA/RNA hybrid analog of the P5 promoter region containing a nascent RNA transcript. A model is proposed for YY1-directed, TBP-independent transcription initiation.
Assuntos
DNA Viral/química , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Proteínas de Ligação a DNA/isolamento & purificação , Dependovirus/genética , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Fator de Transcrição YY1 , Dedos de ZincoRESUMO
Apoptosis has been implicated in ischemic heart disease, but its mechanism in cardiomyocytes has not been elucidated. In this study, we investigate the effects of hypoxia and reoxygenation in adult cardiomyocytes and the molecular mechanism involved in cardiomyocyte apoptosis. Morphologically, reoxygenation induced rounding up of the cells, appearance of membrane blebs that were filled with marginated mitochondria, and ultrastructural findings characteristic of apoptosis. Reoxygenation (18 hours of reoxygenation after 6 hours of hypoxia) and prolonged hypoxia (24 hours of hypoxia) resulted in a 59% and 51% decrease in cellular viability, respectively. During reoxygenation, cell death occurred predominantly via apoptosis associated with appearance of cytosolic cytochrome c and activation of caspase-3 and -9. However, nonapoptotic cell death predominated during prolonged hypoxia. Both caspase inhibition and Bcl-2 overexpression during reoxygenation significantly improved cellular viability through inhibition of apoptosis but had minimal effect on hypoxia-induced cell death. Bcl-2 overexpression blocked reoxygenation-induced cytochrome c release and activation of caspase -3 and -9, but caspase inhibition alone did not block cytochrome c release. These results suggest that apoptosis predominates in cardiomyocytes after reoxygenation through a mitochondrion-dependent apoptotic pathway, and Bcl-2 prevents reoxygenation-induced apoptosis by inhibiting cytochrome c release from the mitochondria and prevents activation of caspase-3 and -9.
Assuntos
Apoptose/fisiologia , Hipóxia Celular/fisiologia , Coração/fisiologia , Miocárdio/citologia , Aerobiose , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Inibidores de Caspase , Grupo dos Citocromos c/metabolismo , Feminino , Genes bcl-2 , Humanos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Csx/Nkx2.5 is an evolutionarily conserved homeodomain (HD)-containing transcription factor that is essential for early cardiac development. We found that the HD of Csx/Nkx2.5 binds as a monomer as well as a dimer to its DNA binding sites in the promoter of the atrial natriuretic factor (ANF) gene, an in vivo target gene of Csx/Nkx2.5. Csx/Nkx2.5 physically interacts with each other in vitro as well as in cells, and the HD is critical for homodimerization. Lys(193) and Arg(194), located at the COOH-terminal end of HD, are essential for dimerization. Lys(193) is also required for a specific interaction with the zinc finger transcription factor GATA4. Csx/Nkx2.5 can heterodimerize with other NK2 homeodomain proteins, Nkx2.3 and Nkx2.6/Tix, with different affinities. A single missense mutation, Ile(183) to Pro in the HD of Csx/Nkx2.5, preserved homodimerization function, but totally abolished DNA binding. Ile(183) --> Pro mutant acts in an inhibitory manner on wild type Csx/Nkx2.5 transcriptional activity through the ANF promoter in 10T1/2 cells. However, Ile(183) --> Pro mutant does not inhibit wild type Csx/Nkx2.5 function on the ANF promoter in cultured neonatal cardiac myocytes, possibly due to failure of dimerization in the presence of the target DNA. These results suggest that complex protein-protein interactions of Csx/Nkx2.5 play a role in its transcriptional regulatory function.
Assuntos
Proteínas de Homeodomínio/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus , Animais , Animais Recém-Nascidos , Arginina/metabolismo , Fator Natriurético Atrial/genética , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator de Transcrição GATA4 , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Ratos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
A new P1O1R9 inducible promoter (where P1 is a promoter sequence analogous to that of the phage T5 early promoter; O1 is lac-operator; and R9 is a ribosome binding site) was synthesized. We studied the efficiency of this promoter in controlling and inducible gene expression using two model genes: human calcitonin tetrameric (hCT[4]) and human interferon alpha 1 (hIFN alpha 1). The synthetic lac-operator O1 was found to repress P1 activity in media free of lac-operon inducers which was derepressed in the presence of IPTG. The levels of expression of both genes (evaluated by quantitating mRNA and protein) in the presence of lac-inducers were close to those obtained with the P1R9 constitutive promoter.
Assuntos
Calcitonina/genética , Regulação Viral da Expressão Gênica , Interferon Tipo I/genética , Regiões Promotoras Genéticas , Fagos T/genética , Sequência de Bases , Clonagem Molecular , Humanos , Cinética , Dados de Sequência MolecularRESUMO
p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , TATA Box , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas Fúngicas/metabolismo , Genes Supressores de Tumor , Humanos , Técnicas In Vitro , Ligação Proteica , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Proteína de Ligação a TATA-BoxRESUMO
Overexpression of the transcription factor YY1 activates DNA synthesis in differentiated primary human coronary artery smooth muscle cells. Overexpression of the retinoblastoma protein together with YY1 blocked this effect. In growth-arrested cells, YY1 resides in a complex with the retinoblastoma protein, but the complex is not detected in serum-stimulated S phase cultures, indicating that the interaction of the retinoblastoma protein and YY1 is cell cycle-regulated. Recombinant retinoblastoma protein directly interacts with YY1, destabilizing the interaction of YY1 with DNA and inhibiting its transcription initiator function in vitro. We conclude that in differentiated cells elevation of the nuclear level of YY1 protein favors progression into the S phase, and we propose that this activity is regulated by its interaction with the retinoblastoma protein.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteína do Retinoblastoma/fisiologia , Fatores de Transcrição/fisiologia , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular , DNA/biossíntese , DNA/metabolismo , Proteínas de Ligação a DNA/química , Fatores de Ligação de DNA Eritroide Específicos , Proteína do Retinoblastoma/química , Fatores de Transcrição/química , Transcrição Gênica , Fator de Transcrição YY1RESUMO
The crystal structure of the transcription factor IIB (TFIIB)/TATA box-binding protein (TBP)/TATA-element ternary complex is described at 2.7 A resolution. Core TFIIB resembles cyclin A, and recognizes the preformed TBP-DNA complex through protein-protein and protein-DNA interactions. The amino-terminal domain of core TFIIB forms the downstream surface of the ternary complex, where it could fix the transcription start site. The remaining surfaces of TBP and the TFIIB can interact with TBP-associated factors, other class II initiation factors, and transcriptional activators and coactivators.